Trixylyl phosphate

Biodegradation in water and sediment: simulation tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: simulation testing on ultimate degradation in surface water

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 26, 2017 to January 17, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study, requested as part of substance evaluation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 309 (Aerobic Mineralisation in Surface Water - Simulation Biodegradation Test)

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

other: Tuckahoe Lake, Queen Anne MD

Details on source and properties of surface water:

The water used in this study was collected from Tuckahoe Lake, Queen Anne MD on October 25, 2017.
The test system was selected to be consistent with study guidelines. The water was collected from the surface (air:water interface). The water temperature at collection was 18.1 °C, the pH was 7.1, and the dissolved oxygen (DO) level was 7.97 mg/L. The water was stored refrigerated prior to dispensing for use.
The water was filtered through a 100 μm filtration system consisting of a 100 μm woven mesh filter and a plug of glass wool prior to characterization and use. The water was clear and colorless before and after filtration.
In addition to the temperature, pH, DO, and appearance, the following characteristics of the water were determined: total nitrogen, ammonium, nitrite, nitrate, total phosphorus, dissolved orthophosphate, total organic carbon, dissolved organic carbon, biochemical oxygen demand (BOD), and total suspended solids.

Details on source and properties of sediment:

Not applicable

Details on inoculum:

Not applicable

Duration of test (contact time):

60 d

Initial conc.:

10 µg/L

Based on:

other: 14C

Initial conc.:

220 µg/L

Based on:

other: 14C

Parameter followed for biodegradation estimation:

radiochem. meas.

Details on study design:

Stock Solution Preparation
A primary stock solution of [14C] Trixylyl Phosphate was prepared by dissolving the entire quantity received in 4 mL of acetonitrile (ACN). The stock solution was identified as 13946-001, and had nominal concentrations of 1,054,500 DPM/μL, or 4.85 μg/μL. An aliquot of the stock solution was diluted 1:100 with ACN and triplicate samples of the dilution were taken for LSC analysis. The mean measured amount of 14C in the primary stock solution was 934,567 DPM/μL (4.30 μg/μL), or 88.6% of the nominal concentration. The remaining stock solution was stored in a freezer when not in use, and was used to prepare dose solutions and HPLC standards for the study.
 
A primary stock solution of the [14C] benzoic acid reference substance was prepared by dissolving the entire quantity received in 20 mL of ethanol. The stock solution was identified as 12190-001, and had nominal concentrations of 111,000 DPM/μL, or 0.081 μg/μL. Triplicate aliquots of the stock solution were taken for LSC analysis, and the mean measured amount of 14C was 102,267 DPM/μL (0.075 μg/μL), or 92.1% of nominal. The stock solution was stored in a refrigerator when not in use, and was used to dose the vessels assigned to the reference substance treatment group.
 
Working Solution Preparation
A working solution of [14C] Trixylyl Phosphate was prepared by combining 200 μL of the primary stock 13946-001 with 285 μL of ACN. This solution was identified as 13946-072517, and had nominal concentrations of 385,388 DPM/μL, or 1.77 μg/μL. For LSC analysis, a dilution of the working stock was prepared by combining 10 μL of 13946-072517 with 990 μL of ACN. This solution was identified as 13946-072517-1 (100X), and had a nominal concentration of 3854 DPM/μL. The measured
concentration of the diluted working stock 13946-072517-1 (100X) was 3647 DPM/μL, which equated to a measured concentration in the working stock 13946-072517 of 364,727 DPM/μL (1.68 μg/μL), or 94.6% of nominal. The working stock was stored in a freezer when not in use, and the 100X dilution was stored in a refrigerator.
 
Dose Solution Preparation
Dose solutions of [14C] Trixylyl Phosphate were prepared on October 27, 2017 by diluting aliquots of the primary stock solution 13946-001 with ACN.
The dose solution for the 10 μg/L treatment group was prepared by diluting 33 μL of the primary stock in 2000 μL of ACN. This solution was identified as 13946-102717-10, and had a nominal concentration of 15170 DPM/μL, or 0.07 μg/μL. Triplicate aliquots of the solution were taken for LSC analysis, and an aliquot was removed for HPLC/β-RAM analysis. The mean measured concentration of the dose solution was 16724 DPM/μL, or 110.2% of nominal, and HPLC/β-RAM analysis indicated that the radiochemical purity of the dose solution was 100%.
 
The dose solution for the 200 μg/L treatment group was prepared by diluting 650 μL of the primary stock in 1350 μL of ACN. This solution was identified as 13946-102717-200, and had a nominal concentration of 303734 DPM/μL, or 1.40 μg/μL. The dose solution was diluted 1:150 in ACN for LSC and HPLC/β-RAM analyses. The mean measured concentration of the dose solution was 345824 DPM/μL, or 113.9% of nominal, and HPLC/β-RAM analysis indicated that the radiochemical purity of
the dose solution was 100%.

Prior to treatment, dose verification solutions were prepared for each [14C] TXP treatment group and the [14C] benzoic acid control group by applying aliquots of the dose solutions to 70 mL of ACN. Based on initial analyses of the dose solutions, the target application volumes were 9.5 μL and 9.0 μL for the low and high level [14C] TXP groups, respectively, and 10 μL for the [14C] benzoic acid group. Aliquots of the dose verification solutions were taken for LSC analysis, and recoveries were 126% and 115% of nominal for the low and high level [14C] TXP groups, respectively, and 107% for the [14C] benzoic acid group.
 
Application of the Test and Reference Substances
Based on the dose verification analyses, the target dose volumes were adjusted to 8.5 μL for the [14C] TXP treatment groups and 8.0 μL for the [14C] benzoic acid group. Aliquots of the dose solutions were measured out using glass 25-microliter syringes, the solutions were applied to the water layers, the vessels were gently swirled to mix the water layers and test substance, then the vessels were sealed with butyl rubber stoppers and crimp caps.
 
Following treatment, aliquots of the dose solutions were analyzed by LSC. Measured concentrations ranged from 112% to 130% of nominal concentrations. Applied doses were calculated based on post-dose solution counts. Actual achieved dose concentrations were 10 μg/L and 220 μg/L for the low and high level [14C] TXP treatment groups, respectively, and 10 μg/L for the [14C] benzoic acid treatment group.
 
Test Conditions
After treatment, all vessels, with the exception of those scheduled for analysis on Day 0, were placed in a temperature controlled incubator set to maintain 12 °C, on a reciprocal shaker table set at approximately 150 oscillations/minute (osc/min). The temperature in the incubator was monitored continuously using an automated environmental monitoring system (AmegaView, version 3.0, Amega Scientific Corporation, Marlton NJ), along with the incubator’s internal monitoring system.
 
Characterization Measurements
Negative and solvent control vessels were sacrificed for characterization measurements (pH and dissolved oxygen [DO]) on each [14C] TXP sampling day.
 
Sampling and Sample Processing
Duplicate [14C] TXP-treated vessels were removed from incubation on Days 0, 7, 10, 21, 35 and 60. The two [14C] benzoic acid-treated vessels were removed on Days 7, 21, 28, and 35. Upon removal from the incubator, vessels were attached to an air flow-through system designed to purge the vessel headspace and trap 14CO2 and 14C volatile gases (Figure 1). The vessels’ septa were pierced with hypodermic needles in two places to provide intake and outflow ports. The intake port was open to room air via connection to a flowmeter in order to monitor airflow. The outflow port was connected to a series of gas trapping vials. Effluent from each test vessel was drawn through the trapping vials via a vacuum source. The trapping train consisted of four 40-mL vials. The first vial was empty to act as a security
trap. The second vial contained 25 mL of ethylene glycol (EG) to trap 14C volatile gases. The third vial was empty to act as another security trap, and the fourth vial contained 25 mL of 1.5 M potassium hydroxide (KOH) to trap 14CO2. The KOH trap was connected to a vacuum manifold to draw air through the test vessel and traps. The vacuum manifold was equipped with individual valves for each test vessel.
 
Connections were made with tygon and teflon tubing. Sterile filters were attached to the intake line of each of the sterilized vessels during purging. Air flow was typically set to maintain a flow rate of approximately 10 mL/min. to produce slow, steady bubbling in the trap vials. Vessel headspaces were purged with at least 5 headspace-volumes of air. Three replicate 1-mL aliquots of each EG and KOH trap were taken for LSC analysis.
 
After purging, the vessels were removed from the trapping system. The [14C] benzoic acid group vessels were sealed with fresh septa and crimp caps and returned to incubation, while the [14C] TXP treated vessels were processed for analyses. The contents of each vessel were extracted with acetonitrile.
Approximately 70 mL of ACN was added to each vessel, the vessels were sonicated for at least 10 minutes, then were placed on a shaker table and shaken at ~120 rpm for at least one hour. The extract volumes were measured, the extracts were transferred to glass bottles, and three replicate 1-mL aliquots were taken for LSC analysis.
 
At the Day 7 analysis, there was a significant deficit in material balances for both treatment groups (10 and 200 μg/L), and it was suspected that some of the test material may have adsorbed to the butyl rubber septa via splashing while being agitated on the shaker table. The septa were disposed of before this could be investigated; therefore, on Day 10, supplemental extractions were conducted on the septa that had been on the vessels during incubation, to try and recover any [14C] TXP that had potentially been adsorbed. The septa were placed in glass bottles, and ~30-mL aliquots of methanol (MeOH) were added to each one. The bottles were sonicated for at least 5 minutes, the extract volumes were measured, and aliquots were taken for LSC analysis. The extraction procedure was then repeated for each septum with ~30 mL of tetrahydrofuran (THF). The methanol extractions recovered only about 6% AR, and were not evaluated further, while the THF extractions recovered >73% AR. Therefore, THF was selected as the solvent of choice for future cap extractions. On Day 21, the septa were first extracted by placing them in the test vessel along with the CAN water mixture to see if adequate recoveries could be obtained with combined extraction, but any additionally recovered TXP was negligible. Therefore, the caps were subjected to a separate extraction with THF, as described above.
 
At each sampling interval after Day 21, the water and septa were processed separately; the water was extracted with ACN as described above, while the septa were extracted with THF in order to recover any potentially adsorbed [14C] TXP.
 
Radiochemical Analyses
The amounts of radioactivity in samples were measured using a Perkin-Elmer Tri-Carb 2900TR or 2910TR Liquid Scintillation Analyzer (LSC) running QuantaSmartTMsoftware (version 2.03 or 4.00).
 
Measured aliquots of each sample were added to scintillation vials containing Ultima Gold XR (Perkin-Elmer) scintillation cocktail. Each group of samples was preceded by one background determination, and the background was subtracted from each sample. The counts were corrected for quench by comparison to known standards using the transformed spectral index of the external standards (tSIE) quench curve.
 
Triplicate aliquots of each sample were counted for at least two minutes. The limit of detection (LOD) of the scintillation counter was estimated to be approximately 25 DPM (typical 14C background during instrument verification). Samples that contained radioactivity above the background level were quantitated. The limit of quantitation (LOQ) of the scintillation counter was the same as the LOD, and was equivalent to 0.1150 ng of Trixylyl Phosphate, equivalent to 0.0164% of applied radioactivity (AR) for the low dose, and 0.0007% AR for the high dose.
 
The LOQ was calculated for the various samples analyzed in the study, based on the applied dosages of 152,126 DPM (0.07 μCi) for the 10 μg/L treatment group and 3,345,039 DPM (1.51 μCi) for the 200 μg/L group. Calculations were based on typical sample volumes and the sizes of the aliquots removed for LSC analyses. The LOQ for the water extracts was approximately 2.3% AR for the low dose group and approximately 0.10% AR for the high dose group. The LOQ for the septa cap extracts was approximately 0.49% AR for the low dose group and approximately 0.02% AR for the high dose group. The LOQ for the ethylene glycol and KOH solutions in the gas traps was approximately 0.4% of the applied dose for the low dose group and approximately 0.02% of the applied dose for the high dose group.
 
Material Balance Analyses
The total radioactivity in each analyzed component was measured and expressed as the percent of applied radioactivity (% of Dose). The radioactivity in the water extracts plus the amounts of 14C recovered from the gas traps, and when applicable, septum cap extractions, were used to determine the radioactive material balance (recovery) for each test vessel.
 
Analytical Method Development
Analytical methods were developed and verified at EAG Laboratories, Easton, prior to the start of the study in a non-GLP method development project. Details of the method development work are documented and filed under Project Number 616E-119 in the archives located on the EAG Laboratories- Easton site. Water samples were fortified with [14C] TXP and ACN was used to extract the samples.
 
Calculations
Results were calculated using Microsoft Excel 2010 in full precision mode. The values presented in the tables and appendices of the report were rounded. Manual calculations using the rounded values may produce slightly different results.
 
For each sample analyzed by LSC, the mean DPM/mL was calculated from the measured
radioactivity in each aliquot and the aliquot size. The sample mean was multiplied by the volume to determine the total radioactivity (Total DPM) in each sample. The total radioactivity was divided by the amount of applied radioactivity to determine the percent of applied radioactivity (% AR) in each sample.
 
The percentages of applied radioactivity in the water extracts plus the amounts in the traps, and when applicable, amounts from septum cap extractions were used to determine the radioactive material balance for each test vessel.
 
For each sample analyzed by HPLC/β-RAM, the parent and any 14C product peaks were integrated from the β-RAM signal using the same parameters in ChemStation Software. The integrated peak areas were assigned to regions of interest (ROI) based on retention times. The peak areas within each region were divided by the total peak area of each sample to determine the percent of 14C in each region. The percent of 14C in each region was multiplied by the “%AR” to determine the relative distribution of radioactivity within each sample.

Reference substance:

other: Benzoic Acid [ring-14C(U)]

Test performance:

Temperatures in the incubator ranged from 11.7 to 14.6 degrees C during the 60-day incubation period, and the average temperature was 12.0 °C. Minor excursions from the 12 ± 2 °C nominal range occurred, primarily during opening of the incubator for study-related activities. These minor variations had no impact on study results.

Key result

Compartment:

water

DT50:

> 60 d

Type:

(pseudo-)first order (= half-life)

Temp.:

12 °C

Key result

Compartment:

water

DT50:

> 60 d

Type:

other: DT90

Temp.:

12 °C

Transformation products:

no

Details on transformation products:

Transformation Results in Live Vessels
No non-parent peaks were integrated in any of the chromatograms of water samples. All radioactivity was accounted for by parent test substance.

A minor non-parent peak was present in one sample from the Day 35 interval, and in both samples from the Day 60 interval. All these peaks appeared in the same ROI, eluting immediately after the parent. The maximum amount of radioactivity in any individual peak was 3.3% AR, and the maximum mean amount was 2.5% AR, occurring at Day 60.

At 60 days, parent material accounted for 91.7% AR, and transformation products accounted for 2.5% AR. Since the transformation products were present at <10%, they were not defined as major transformation products based on OECD guidelines.

Transformation Results in Sterile Vessels
No non-parent peaks were detected in any of the sterile vessel extracts analyzed.

Evaporation of parent compound:

not specified

Volatile metabolites:

no

Residues:

not specified

Details on results:

Test Conditions
Temperatures in the incubator ranged from 11.7 to 14.6 degrees C during the 60-day incubation period, and the average temperature was 12.0 °C. Minor excursions from the 12 ± 2 °C nominal range occurred, primarily during opening of the incubator for study-related activities. These minor variations had no impact on study results.

Water Characterization
Characterization test vessels were sacrificed for measurements on Days 0, 7, 10, 21, 35 and 60. The pH ranged from 6.65 to 7.61, while DO ranged from 7.84 to 10.22 mg/L, and was above the minimum acceptable level (2.0 mg/L) during the 60-day incubation period.

Live Transformation Vessel Gas Trap Results
14C gas production was minimal in both test groups over the course of the study. At Day 60, total gas production was 0.48% AR in the 10 μg/L group and 0.14% AR in the 200 μg/L group.

Sterile Transformation Vessel Gas Trap Results
The LSC results from traps collected from the sacrificed sterile transformation test vessels at the final sampling interval. Total gas production over the 60-day test duration was 0.01% AR in the sterile vessels.

Material Balance Results, Live Vessels
On Day 0, mean material balances were 111.8% AR in the 10 μg/L group, and 97.1% AR in the 200 μg/L group. On Day 7, there was a significant deficit in material balances for both groups, likely attributable to adsorption of TXP to the butyl rubber septa used to seal the test vessels. On Day 10 and beyond, the septa were extracted as well, and thereafter, mean material balances ranged from 105.1% AR to 121.5% AR for the 10 μg/L group, and from 91.7% AR to 97.4% AR for the 200 μg/L group.

Material Balance Results, Sterile Vessels
The mean material balance was 98.3% AR on Day 0, and 96.5% AR on Day 60.

Results with reference substance:

Reference Substance Vessel Gas Trap Results
Mean cumulative 14C gas production reached >60% by Day 28, and both vessels exceeded the 60% acceptance criteria by Day 35, indicating that the microbial population was viable over the course of the study.

Water ID: TL-102517 Properties

Date collected: Oct. 25, 2017

Location: Tuckahoe Lake, Queen Anne MD

Location coordinates: Lat. 38.9686’ Long. -75.9447’

Collection Depth: Surface; Air:water interface

Performed by EAG Laboratories, Easton

Temperature at collection: 18.1 °C

pH at collection: 7.1

DO at collection: 7.97 mg/L

Appearance: Clear/colorless

Performed by Agvise Laboratories, Northwood, ND

Total Nitrogen: 4.6 ppm

Ammonium Nitrogen: 0.4 ppm

Nitrate: 4.6 ppm

Nitrite: <0.1 ppm

Total Phosphorus: 0.6 ppm

Dissolved orthophosphate: 0.6 ppm

Total organic carbon: 2.2 ppm

Dissolved organic carbon: 1.9 ppm

Suspended solids: 8 ppm

Biological Oxygen Demand: 0.6 ppm

Water Characterization Results

Sample Interval	Control Group ID	pH	DO (mg/L)
Day 0	Blank	7.25	9.87
	Solvent	7.39	10.06
Day 7	Blank	7.43	10.22
	Solvent	7.61	10.12
Day 10	Blank	6.76	9.84
	Solvent	7.12	9.79
Day 21	Blank	7.54	9.94
	Solvent	7.26	9.28
Day 35	Blank	6.65	10.03
	Solvent	7.16	7.90
Day 60	Blank	6.92	10.00
	Solvent	6.93	7.84

Measured Radioactivity in Vessel Gas Traps (%AR), Live Vessels, 10 μg/L

 

Interval (days)	Vessel ID	Volatile 14C Gases	Mean	Evolved 14CO2	Mean	Total Gases	Mean
7	3	0.00	0.13	0.01	0.00	0.01	0.13
	4	0.25		0.00		0.25
10	13	0.00	0.02	0.05	0.08	0.05	0.10
	14	0.03		0.10		0.14
21	5	0.14	0.18	0.11	0.09	0.25	0.27
	6	0.22		0.07		0.28
35	7	0.01	0.03	0.17	0.24	0.18	0.27
	8	0.05		0.31		0.36
60	9	0.03	0.12	0.11	0.37	0.14	0.48
	10	0.20		0.62		0.82

 

Measured Radioactivity in Vessel Gas Traps (%AR), Live Vessels, 200 μg/L

Interval (days)	Vessel ID	Volatile 14C Gases	Mean	Evolved 14CO2	Mean	Total Gases	Mean
7	19	0.01	0.01	0.01	0.01	0.02	0.02
	20	0.01		0.01		0.02
10	29	0.03	0.02	0.00	0.01	0.03	0.03
	30	0.01		0.02		0.03
21	21	0.01	0.01	0.06	0.06	0.07	0.07
	22	0.00		0.07		0.07
35	23	0.00	0.00	0.11	0.11	0.11	0.11
	24	0.00		0.12		0.12
60	25	0.01	0.01	0.16	0.13	0.17	0.14
	26	0.01		0.10		0.11

 

Measured Radioactivity in Vessel Gas Traps (%AR), Sterile Vessels, 200 μg/L

Interval (days)	Vessel ID	Volatile 14C Gases	Mean	Evolved 14CO2	Mean	Total Gases	Mean
60	35	0.01	0.00	0.00	0.00	0.01	0.01
	36	0.00		0.00		0.00

 

Measured Radioactivity in Reference Vessel (Benzoic Acid) Gas Traps (%AR)

Interval (days)	Vessel ID	Volatile 14C Gases	Mean	Evolved 14CO2	Mean	Total Gases	Mean
7	39	0.04	0.03	14.67	18.36	14.71	18.39
	10	0.02		22.06		22.08
21	39	0.28	0.28	32.17	33.22	32.45	33.50
	10	0.28		34.27		34.55
28	39	0.13	0.14	12.65	15.12	12.79	15.26
	10	0.14		17.59		17.73
35	39	0.08	0.27	5.80	9.84	5.88	10.11
	10	0.47		13.87		14.34
Cumulative	39	0.53	0.72	65.79	76.54	65.83	77.26
	10	0.90		87.79		88.69

 1 Sampling terminated after Day 35; mineralization criteria met (>60% AR)

 

Distribution of Radioactivity from Sacrificed Test Vessels (%AR), Live Vessels, 10 μg/L

Interval (days)	Vessel ID	Water extracts	Mean	Cap Extracts	Mean	Total Gases	Mean	Material Balance (Recovery)	Mean Material Balance
0	1	111.6	111.8	NA	NA	NA	NA	111.6	111.8
	2	112.0		NA		NA		112.0
7	3	12.2	11.5	NA	NA	0.01	0.13	12.2	11.7
	4	10.9		NA		0.25		11.1
10	13	15.2	12.2	92.6^1	92.8	0.05	0.10	107.8	105.1
	14	9.2		93.0^2		0.14		102.4
21	5	22.2	23.5	97.4	93.1	0.25	0.27	119.9	116.8
	6	24.7		88.8		0.28		113.8
35	7	7.9	6.5	100.8	102.5	0.18	0.27	108.9	109.2
	8	5.0		104.2		0.36		109.5
60	9	12.8	13.5	109.7	107.5	0.14	0.48	122.7	121.5
	10	14.1		105.4		0.82		120.3

NA – Not Applicable

1 Includes 86.5% AR from THF extract and 6.0% AR from MeOH extract

2 Includes 87.1% AR from THF extract and 6.0% AR from MeOH extract

Distribution of Radioactivity from Sacrificed Test Vessels (%AR), Live Vessels, 200 μg/L

Interval (days)	Vessel ID	Water extracts	Mean	Cap Extracts	Mean	Total Gases	Mean	Material Balance (Recovery)	Mean Material Balance
0	17	97.0	97.1	NA	NA	NA	NA	97.0	97.1
	18	97.2		NA		NA		97.2
7	19	34.3	30.7	NA	NA	0.02	0.02	34.3	30.8
	20	27.1		NA		0.02		27.2
10	29	10.7	12.1	79.9^1	81.4	003	0.03	90.6	93.5
	30	13.6		82.8^2		0.03		96.4
21	21	20.2	19.9	76.1	77.4	0.07	0.07	96.4	97.4
	22	19.5		78.7		0.07		98.3
35	23	4.9	5.2	84.0	86.4	0.11	0.11	89.0	91.7
	24	5.5		88.8		0.12		94.4
60	25	8.0	7.9	89.8	86.3	0.17	0.14	97.9	94.4
	26	7.8		82.9		0.11		90.8

NA – Not Applicable

1 Includes 73.8% AR from THF extract and 6.1% AR from MeOH extract

2 Includes 76.6% AR from THF extract and 6.2% AR from MeOH extract

 

Distribution of Radioactivity from Sacrificed Test Vessels (%AR), Sterile Vessels, 200 μg/L

Interval (days)	Vessel ID	Water extracts	Mean	Cap Extracts	Mean	Total Gases	Mean	Material Balance (Recovery)	Mean Material Balance
0	33	97.2	98.3	NA	NA	NA	NA	97.2	98.3
	34	99.5		NA		NA		99.5
60	35	5.7	5.5	92.8	91.0	0.01	0.01	98.5	96.5
	36	5.4		89.2		0.00		94.6

NA – Not Applicable

 

Distribution of [14C] Trixylyl Phosphate Components in ACN Water Extracts (%AR)

Treatment	Interval (days)	Vessel ID	TXP	Mean	ROI 2	Mean	Total	Mean	Transf. Products	Mean
Live200 μg/L	0	17	97.0	97.1	0.0	0.0	97.0	97.1	0.0	0.0
		18	97.2		0.0		97.2		0.0
	7	19	3433	30.7	0.0	0.0	34.3	30.7	0.0	0.0
		20	27.1		0.0		27.1		0.0
	10	19	10.7	12.1	0.0	0.0	10.7	12.1	0.0	0.0
		30	13.6		0.0		13.6		0.0
	21	21	20.2	19.9	0.0	0.0	20.2	19.9	0.0	0.0
		22	19.5		0.0		19.5		0.0
	35	23	4.9	5.2	0.0	0.0	4.9	5.2	0.0	0.0
		24	5.5		0.0		5.5		0.0
	60	25	8.0	7.9	0.0	0.0	8.0	7.9	0.0	0.0
		26	7.8		0.0		7.8		0.0
Sterile200 μg/L	0	33	97.2	98.3	0.0	0.0	97.2	98.3	0.0	0.0
		34	99.5		0.0		99.5		0.0
	60	35	5.7	5.5	0.0	0.0	5.7	5.5	0.0	0.0
		36	5.4		0.0		5.4		0.0

 

Distribution of [14C] Trixylyl Phosphate Components in THF Cap Extracts (%AR)

Treatment	Interval (days)	Vessel ID	TXP	Mean	ROI 2	Mean	Total	Mean	Transf. Products	Mean
Live200 μg/L	0	17	NA	NA	NA	NA	NA	NA	NA	NA
		18	NA		NA		NA		NA
	7	19	NA	NA	NA	NA	NA	NA	NA	NA
		20	NA		NA		NA		NA
	10	19	73.8^1	75.2	0.0	0.0	73.8^1	75.2	0.0	0.0
		30	76.6^1		0.0		76.6^1		0.0
	21	21	76.1	77.4	0.0	0.0	76.1	77.4	0.0	0.0
		22	78.7		0.0		78.7		0.0
	35	23	84.0	84.7	0.0	1.6	84.0	84.7	0.0	1.6
		24	85.5		3.3		85.5		3.3
	60	25	87.7	83.9	2.1	2.5	87.7	83.9	2.1	2.5
		26	80.0		2.9		80.0		2.9
Sterile200 μg/L	0	33	NA	NA	NA	NA	NA	NA	NA	NA
		34	NA		NA		NA		NA
	60	35	92.8	91.0	0.0	0.0	92.8	91.0	0.0	0.0
		36	89.2		0.0		89.2		0.0

NA = Not Applicable (not analyzed)

1MeOH cap extracts not analyzed, low radioactivity

 

Distribution of [14C] Trixylyl Phosphate Components in Total System (%AR)

Treatment	Interval (days)	Vessel ID	TXP	Mean	ROI 2	Mean	Total	Mean	Transf. Products	Mean
Live200 μg/L	0	17	97.0	97.1	0.0	0.0	97.0	97.1	0.0	0.0
		18	97.2		0.0		97.2		0.0
	7	19	34.3	30.7	0.0	0.0	34.3	30.7	0.0	0.0
		20	27.1		0.0		27.1		0.0
	10	19	84.5	87.3	0.0	0.0	84.5	87.3	0.0	0.0
		30	90.1		0.0		90.1		0.0
	21	21	96.3	87.3	0.0	0.0	96.3	97.3	0.0	0.0
		22	98.2		0.0		98.2		0.0
	35	23	88.9	97.3	0.0	1.6	88.9	91.6	0.0	1.6
		24	91.0		3.3		94.3		3.3
	60	25	95.7	89.9	2.1	2.5	97.8	94.2	2.1	2.5
		26	87.8		2.9		90.7		2.9
Sterile200 μg/L	0	33	97.2	91.7	0.0	0.0	97.2	98.3	0.0	0.0
		34	99.5		0.0		99.5		0.0
	60	35	98.5	96.5	0.0	0.0	98.5	96.5	0.0	0.0
		36	94.6		0.0		94.6		0.0

 

Validity criteria fulfilled:

yes

Conclusions:

Mean material balances ranged from 105.1% AR to 121.5% AR for the 10 μg/L group, with the exception of Day 7, when the septa caps were not extracted, and from 91.7% AR to 97.4% for the live 200 μg/L group, again with the exception of Day 7.

Mean material balances for the 200 μg/L sterile group were 98.3% AR on Day 0 and 96.5% AR on Day 60.

Mineralization was minimal throughout the course of the study, with 14C gas production of 0.48% AR in the 10 μg/L group , 0.14% AR in the live 200 μg/L group, and 0.01% AR in the sterile 200 μg/L group.

Mineralization in the reference substance group reached >60% by Day 28, and both vessels exceeded the 60% acceptance criteria by Day 35, indicating that the microbial population was viable over the course of the study.

HPLC/β-RAM analyses of samples from the live and sterile 200 μg/L treatment groups showed that the parent compound Trixylyl Phosphate remained substantially intact in all live and sterile vessels throughout the course of this mineralization in aerobic water study. No non-parent peaks were detected in any of the sterile vessel samples, or in any of the ACN water extract samples from the live vessels. Minor non-parent peaks were detected in a few of the THF cap extract samples from the live vessels at Days 35 and 60, and these accounted for mean maximum of 2.5% AR at Day 60. Since the transformation products were present at <10%, they were not defined as major transformation products based on OECD guidelines.

The DT50 and DT90 values are reported as >60 days, the duration of the study.

Executive summary:

This study was conducted to assess the potential mineralization and transformation of Trixylyl Phosphate in aerobic water systems. Natural lake water was utilized in the study. Water was dosed with 14C-labeled Trixylyl Phosphate at target concentrations of ~10 μg/L and ~200 μg/L (actual concentrations were 10 μg/L and 220 μg/L). Sterilized water dosed at 220 μg/L was included to evaluate transformation in the absence of water microorganisms. Additional vessels were dosed with 14C benzoic acid at a target concentration of ~10 μg/L (actual concentration was 10 μg/L) to assess viability of the microbial population. After dosing, test vessels were sealed with septa and crimp caps, and were incubated at approximately 12 ºC for up to 60 days with continuous shaking except for brief periods during study related activities requiring handling or observation of vessels. Aerobic conditions were monitored by

dissolved oxygen measurements in control vessels. At each sampling interval, vessels selected for analysis were attached to an air flow-through system designed to purge vessel headspace with air and collect 14C volatile gases and 14CO2. Effluent gases were passed through ethylene glycol to trap organic volatiles, followed by an alkali solution to trap evolved carbon dioxide. Duplicate live test vessels of each dose level were sacrificed on Days 0, 7, 10, 21, 35 and 60. Duplicate sterile vessels were sacrificed on Days 0 and 60. Reference substance (benzoic acid) vessels were assessed for 14C gas production on Days 7, 21, 28 and 35. Water samples were extracted with acetonitrile (ACN) and analyzed for total radioactivity by liquid scintillation counting (LSC). On Days 10 and beyond, the septa caps were extracted with tetrahydrofuran (THF). The mean percent of applied radioactivity recovered is presented in the following table for live vessels:

Nominal test level	Interval (Day)	ACN Water Extracts (%)	THF CAP Extracts (%)	Total gases (%)	Material Balance (Recovery) (%)
10 μg/L	0	111.8	NA	NA	111.8
	7	11.5	NA	0.13	11.7
	10	12.2	92.8	0.10	105.1
	21	23.5	93.1	0.27	116.8
	35	6.5	102.5	0.27	109.2
	60	13.5	107.5	0.48	121.5
200 μg/L	0	97.1	NA	NA	97.1
	7	30.7	NA	0.02	30.8
	10	12.1	81.4	0.03	93.5
	21	19.9	77.4	0.07	97.4
	35	5.2	86.4	0.11	91.7
	60	7.9	86.3	0.14	94.4

NA - Not Applicable

 

The mean percent of applied radioactivity recovered from sterile vessels is presented in the following table:

Nominal test level	Interval (Day)	ACN Water Extracts (%)	THF CAP Extracts (%)	Total gases (%)	Material Balance (Recovery) (%)
200 μg/L	0	98.3	NA	NA	98.3
	60	5.5	91.0	0.01	96.5

 

Mean material balances ranged from 105.1% AR to 121.5% AR for the 10 μg/L group, with the exception of Day 7, when the septa caps were not extracted, and from 91.7% AR to 97.4% for the live 200 μg/L group, again with the exception of Day 7.

 

Mean material balances for the 200 μg/L sterile group were 98.3% AR on Day 0 and 96.5% AR on Day 60.

Mineralization was minimal throughout the course of the study, with 14C gas production of 0.48% AR in the 10 μg/L group, 0.14% AR in the live 200 μg/L group, and 0.01% AR in the sterile 200 μg/L group.

 

Mineralization in the reference substance group reached >60% by Day 28, and both vessels exceeded the 60% acceptance criteria by Day 35, indicating that the microbial population was viable over the course of the study.

 

HPLC/β-RAM analyses of samples from the live and sterile 200 μg/L treatment groups showed that the parent compound Trixylyl Phosphate remained substantially intact in all live and sterile vessels throughout the course of this mineralization in aerobic water study. No non-parent peaks were detected in

any of the sterile vessel samples, or in any of the ACN water extract samples from the live vessels. Minor non-parent peaks were detected in a few of the THF cap extract samples from the live vessels at Days 35 and 60, and these accounted for a mean maximum of 2.5% AR at 60 days.. Since the transformation products were present at <10%, they were not defined as major transformation products based on OECD guidelines.

 

The DT50 and DT90 values are reported as >60 days, the duration of the study.

Description of key information

Mean material balances ranged from 105.1% AR to 121.5% AR for the 10 μg/L group, with the exception of Day 7, when the septa caps were not extracted, and from 91.7% AR to 97.4% for the live 200 μg/L group, again with the exception of Day 7.

Mean material balances for the 200 μg/L sterile group were 98.3% AR on Day 0 and 96.5% AR on Day 60.

Mineralization was minimal throughout the course of the study, with 14C gas production of 0.48% AR in the 10 μg/L group , 0.14% AR in the live 200 μg/L group, and 0.01% AR in the sterile 200 μg/L group.

Mineralization in the reference substance group reached >60% by Day 28, and both vessels exceeded the 60% acceptance criteria by Day 35, indicating that the microbial population was viable over the course of the study.

HPLC/β-RAM analyses of samples from the live and sterile 200 μg/L treatment groups showed that the parent compound Trixylyl Phosphate remained substantially intact in all live and sterile vessels throughout the course of this mineralization in aerobic water study. No non-parent peaks were detected in any of the sterile vessel samples, or in any of the ACN water extract samples from the live vessels. Minor non-parent peaks were detected in a few of the THF cap extract samples from the live vessels at Days 35 and 60, and these accounted for mean maximum of 2.5% AR at Day 60. Since the transformation products were present at <10%, they were not defined as major transformation products based on OECD guidelines.

The DT50 and DT90 values are reported as >60 days, the duration of the study.

Key value for chemical safety assessment

Half-life in freshwater:

60 d

at the temperature of:

12 °C

Additional information

This study was conducted to assess the potential mineralization and transformation of Trixylyl Phosphate in aerobic water systems. Natural lake water was utilized in the study. Water was dosed with 14C-labeled Trixylyl Phosphate at target concentrations of ~10 μg/L and ~200 μg/L (actual concentrations were 10 μg/L and 220 μg/L). Sterilized water dosed at 220 μg/L was included to evaluate transformation in the absence of water microorganisms.

Additional vessels were dosed with 14C benzoic acid at a target concentration of ~10 μg/L (actual concentration was 10 μg/L) to assess viability of the microbial population. After dosing, test vessels were sealed with septa and crimp caps, and were incubated at approximately 12 ºC for up to 60 days with continuous shaking except for brief periods during study related activities requiring handling or observation of vessels. Aerobic conditions were monitored by dissolved oxygen measurements in control vessels. At each sampling interval, vessels selected for analysis were attached to an air flow-through system designed to purge vessel headspace with air and collect 14C volatile gases and 14CO2. Effluent gases were passed through ethylene glycol to trap organic volatiles, followed by an alkali solution to trap evolved carbon dioxide. Duplicate live test vessels of each dose level were sacrificed on Days 0, 7, 10, 21, 35 and 60. Duplicate sterile vessels were sacrificed on Days 0 and 60. Reference substance (benzoic acid) vessels were assessed for 14C gas production on Days 7, 21, 28 and 35. Water samples were extracted with acetonitrile (ACN) and analyzed for total radioactivity by liquid scintillation counting (LSC). On Days 10 and beyond, the septa caps were extracted with tetrahydrofuran (THF). The mean percent of applied radioactivity recovered is presented in the following table for live vessels:

Nominal test level	Interval (Day)	ACN Water Extracts (%)	THF CAP Extracts (%)	Total gases (%)	Material Balance (Recovery) (%)
10 μg/L	0	111.8	NA	NA	111.8
	7	11.5	NA	0.13	11.7
	10	12.2	92.8	0.10	105.1
	21	23.5	93.1	0.27	116.8
	35	6.5	102.5	0.27	109.2
	60	13.5	107.5	0.48	121.5
200 μg/L	0	97.1	NA	NA	97.1
	7	30.7	NA	0.02	30.8
	10	12.1	81.4	0.03	93.5
	21	19.9	77.4	0.07	97.4
	35	5.2	86.4	0.11	91.7
	60	7.9	86.3	0.14	94.4

NA - Not Applicable

 

The mean percent of applied radioactivity recovered from sterile vessels is presented in the following table:

Nominal test level	Interval (Day)	ACN Water Extracts (%)	THF CAP Extracts (%)	Total gases (%)	Material Balance (Recovery) (%)
200 μg/L	0	98.3	NA	NA	98.3
	60	5.5	91.0	0.01	96.5

 

Mean material balances ranged from 105.1% AR to 121.5% AR for the 10 μg/L group, with the exception of Day 7, when the septa caps were not extracted, and from 91.7% AR to 97.4% for the live 200 μg/L group, again with the exception of Day 7.

 

Mean material balances for the 200 μg/L sterile group were 98.3% AR on Day 0 and 96.5% AR on Day 60.

Mineralization was minimal throughout the course of the study, with 14C gas production of 0.48% AR in the 10 μg/L group, 0.14% AR in the live 200 μg/L group, and 0.01% AR in the sterile 200 μg/L group.

 

Mineralization in the reference substance group reached >60% by Day 28, and both vessels exceeded the 60% acceptance criteria by Day 35, indicating that the microbial population was viable over the course of the study.

 

HPLC/β-RAM analyses of samples from the live and sterile 200 μg/L treatment groups showed that the parent compound Trixylyl Phosphate remained substantially intact in all live and sterile vessels throughout the course of this mineralization in aerobic water study. No non-parent peaks were detected in

any of the sterile vessel samples, or in any of the ACN water extract samples from the live vessels. Minor non-parent peaks were detected in a few of the THF cap extract samples from the live vessels at Days 35 and 60, and these accounted for a mean maximum of 2.5% AR at 60 days.. Since the transformation products were present at <10%, they were not defined as major transformation products based on OECD guidelines.

 

The DT50 and DT90 values are reported as >60 days, the duration of the study.